There have been a number of gene transfer vectors that show promise in preclinical studies for hemophilia. To date, there have been 5 gene therapy clinical hemophilia trials using 4 different vectors, and yet none of them have demonstrated sustained clinical efficacy. While one of these vectors, rAAV showed promise in preclinical animals studies, a greater understanding of the potential limitations and safety are required. Nonviral DNA vectors have suffered from two distinct limitations; tissue delivery and the ability to achieve sustained and therapeutic levels of transgene expression. Our laboratory has focused on the later problem and has recently developed mini-circle DNA vectors that remain episomal but result in persistent and therapeutic levels of gene expression in quiescent tissues in vivo. There is renewed interest in episomal vectors because of the recent cases of leukemia in X-linked SCID cases treated with an integrating retroviral vector. Our specific aims are geared towards further understanding the mechanisms of transduction of both of these vectors, their safety, and methods that will contribute to their application of gene therapy for hemophilia and other disorders. Specifically, we will pursue our findings that the efficacy of AAV-mediated gene transduction in the liver is related at least in part to the rapidity of capsid vector uncoating and use this information to make improved vectors for in vivo gene transfer. We plan to establish if other AAV genomes are more robust and have altered mechanisms of transduction, and further establish the oncogenic potential of AAV vectors. For non-viral DNA vectors, we will perform studies that will assist in understanding the mechanism involved in transgene transcriptional silencing of episomal vectors in vivo. To do this, we will unravel the molecular structure of the chromatin formed after plasmid mediated gene transfer in vivo with and without silencing. The studies outlined here will further our understanding of gene transduction and mechanisms of transgene expression as well as have direct applications for gene therapy for hemophilia.